The goal of this project is to test the hypothesis that the tumor microenvironment plays a critical role in influencing T cell responses to tumor antigens. This microenvironment is comprised of a complex interaction between tumor cells, lymphocytes, myeloid cells, and stromal cells. This project uses TRAMP mice which express the transforming SV40 T antigen (TAg) under the transcriptional control of a prostate-specific promoter, which causes the development of murine prostate cancer. Nave TAg-specific T cells are transferred into TRAMP mice. We previously reported that CD8+ T cells become tolerized when they enter the tumor microenvironment. In a report recently published, we identified these tolerant, tumor-specific CD8+ T cells as regulatory, or suppressor, T cells, and induction of suppressor activity is dependent on infiltration into the prostate. These regulatory cells have the capacity to suppress proliferation of other T cells. We also reported that transfer of TAg-specific CD4+ T cells also undergo transient activation before deletion and trafficking to the prostate. Taking advantage of this transient activation, we further demonstrated that co-transfer of both CD4+ and CD8+ T cells delays tolerization of the CD8+ T cells. Continuous administration of the tumor-specific CD4+ T cell prevented T cell tolerance and reduced tumor growth. Our current work is aimed at understanding the mechanism by which T cells become tolerized in the tumor microenvironment. We have identified a population of plasmacytoid dendritic cells (pDCs) which exist in both normal and transformed prostate tissues. Interestingly, the dendritic cells isolated from the TRAMP prostate are incapable of stimulating proliferation of nave T cell proliferation in vitro, whereas dendritic cells from normal prostate tissues are stimulatory. Further examination of the T cells primed by the tumor-associated DC (TADC) revealed that they were tolerized and had suppressor function. Depletion of the TADC resulted in efficient T cell priming and reduced tumor growth. A similar population of pDCs was identified in human prostate cancer specimens, and these cells had similar functional attributes. Most striking was our observation that the transcription factor FOXO3a was over-expressed in TADC from human and murine prostatic tumors (relative to non-cancerous prostatic tissues), and silencing expression of Foxo3a resulted in a loss of TADC tolerogenicity. These findings implicate FOXO3a as a novel target for prostate cancer immunotherapy. Based on these findings, a patent application was submitted in January, 2010. A newer project is focused on studying the role of mast cells in immune regulation in the prostate tumor microenvironment. Mast cells play an important role in allergic responses, but have also been implicated in tumor progression. We have identified a population of tumor-derived mast cells that accumulate in TRAMP tumors as they develop. These mast cells inhibit T cell responses in vitro. Our on-going studies will characterize the mechanisms by which they suppress T cells and identify ways to inhibit this suppression to enhance immunity to prostatic tumors.